During the past year, this laboratory continued studies on the mechanism of HIV entry, as well as development of therapeutic and protective strategies based on the molecules involved in entry. 1) Molecular mechanism of HIV Env-mediated fusion. a. We examined the HIV Env interaction with CD4 and coreceptor in the context of the oligomeric structure of Env. Analyses of the complementing activities of various Env fusion-defective mutants led to the conclusion that a single gp120 molecule must interact with both CD4 and coreceptor; these interactions can then trigger gp41 on another member of the oligomer to induce fusion. 2) Anti-HIV agents based on HIV Env/receptor interactions. a. We studied the molecular mechanism of HIV blockade by the prokaryotic protein cyanovirin-N (CV-N). We found that CV-N strongly inhibits HIV Env-mediated fusion; this activity is mediated by blocking of CD4 binding to gp120, which in turn results in failure of Env to engage coreceptor. We discovered that the antiviral activity of CV-N also extended to feline immunodeficiency virus, measles virus, and human herpesvirsus-6, but not to vaccinia virus. This broader activity of CV-N may suggest its potential use against several virus infections. b. We developed a novel agent which we anticipate will have potent neutralizing activity against genetically diverse HIV-1 genetic subtypes. The agent is a recombinant protein containing soluble CD4 attached via a long flexible polypeptide linker to a single chain antibody that binds to a CD4-induced eptiope of gp120 involved in binding to coreceptor. The plasmid contruct and a corresponding vaccinia construct have been made, with the goal of producing sufficeint quantities of the protein for in vitro and in vivo analysis of anti-HIV activity. c. We have extended our interest in using Env- targeted toxins to help eliminate HIV infection, particularly when used in combination with HAART. We found that the activity of two such toxins, CD4-PE40 and the immunotoxin 3b3-PE38 had efficacy against HIV- 1 acute and chronic infection in SCID-hu mice; the effects were much more pronounced in conjunction with HAART. We have also initiated in vitro and in vivo studies using an HIV transgenic mouse model to study the ability of Env-targeted toxins to block induction of HIV-1 expression by killing the newly activated HIV-expressing cells. Preliminary in vitro results indicate that CD4-PE40 potently kills cells from HIV transgenic mice, and inhibits expression of virus upon cell activation. 3) Vacccine strategy based of Env/CD4/coreceptor interaction. We initiated attempts to isolate hybridomas from mice immunized with ?fusion-competent cells? (mixtures between cells expressing Env and cells expressing CD4 plus coreceptor, J. Nunberg and coworkers). Identification of the epitopes for the broadly cross- neutralizing antibodies generated by this procedure will help in the design of more practical antigens to be used in vaccine formulations. - HIV, Virus Entry, Virus Fusion, Glycoprotein Receptor, Coreceptor, Antivirals?HIV, Neutralization, Env-Targeted Toxin, Vaccines-HIV